Delivery of therapeutic agents by a collagen binding protein

ABSTRACT

Methods of delivering therapeutic agents by administering compositions including a bacterial collagen-binding polypeptide segment linked to the therapeutic agent to subjects in need of treatment with the therapeutic agent are provided. In these methods, the therapeutic agent is not a PTH/PTHrP receptor agonist or antagonist, basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). The bacterial collagen-binding polypeptide segment delivers the agent to sites of partially untwisted or under-twisted collagen. Methods of treating collagenopathies using a composition including a collagen-binding polypeptide and a PTH/PTHrP receptor agonist are also provided. In addition, methods of treating hyperparathyroidism, and hair loss using compositions comprising a collagen binding polypeptide and a PTH/PTHrP receptor agonist are provided. Finally, methods of reducing hair regrowth by administering a composition including a collagen binding polypeptide and a PTH/PTHrP receptor antagonist are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. applicationSer. No. 14/365,226, filed Jun. 13, 2014, which is a national stagefiling under 35 U.S.C. 371 of International Application No.PCT/US2012/069831, filed Dec. 14, 2012, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 61/570,620, filedDec. 14, 2011 and of U.S. Provisional Patent Application No. 61/596,869,filed Feb. 9, 2012, all of which are incorporated herein by reference intheir entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded bythe National Institutes of Health grant number NCRR COBRE 8P30GM103450and INBRE GM103429. The United States may have certain rights in thisinvention.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is incorporatedherein by reference in its entirety. The Sequence Listing was filed withthe application as a text file on Dec. 14, 2012.

INTRODUCTION

Delivery of therapeutic agents to sites within the body of a subjectwhere a particular therapeutic agent is needed in order to be effectiveis a developing area. Such delivery systems will allow more efficientuse of therapeutic agents while reducing toxicity caused by sometherapeutic agents. Use of targeted liposomes or polypeptides, such asantibodies, to target therapeutic agents to particular sites within thebody has proved successful, but additional delivery agents are needed.

Alopecia (hair loss) is a psychologically and emotionally distressingevent with multiple causes. Alopecia occurs most commonly inmale-pattern baldness, affecting approximately two thirds of males byage 35; a similar pattern of hair loss can be observed in females withpolycystic ovarian syndrome. In both of these disorders, the hair lossis androgen mediated. Alopecia can also occur as an autoimmune disease,termed alopecia areata; a disorder which affects 1.7% of the population.It can occur as a side-effect of medical treatments, particularly inchemotherapy, with 65-85% of chemotherapy patients experiencing somedegree of alopecia. Psychological consequences of hair loss have beenwell studied in the chemotherapy setting. Chemotherapy-induced alopecia(CIA) can result in anxiety, depression, a negative body image, loweredself-esteem and a reduced sense of well-being. In fact, 47-58% of femalecancer patients consider hair loss to be the most traumatic aspect ofchemotherapy, and 8% would decline treatment for fear of hair loss. Inaddition to these studies in chemotherapy patients, evidence exists inother forms of alopecia to support therapy to reduce psychologicalconsequences of hair loss. Thus a new treatment to stop hair loss orspeed hair regrowth would be beneficial.

While drugs with mild anti-androgenic effects (i.e. spironolactone) hadbeen used with limited success as therapy for alopecia, the firsteffective medication for alopecia was minoxidil (Rogaine). Thisantihypertensive has an observed side-effect of causing hair growth, andis now used as topical therapy for many forms of alopecia. However,responses are incomplete, with some subjects showing only slowing ofhair loss rather than actual regrowth. Finasteride (Propecia) is a neweragent that blocks conversion of testosterone to dihydrotestosterone,resulting in improvements in androgenic alopecia at the expense ofpartial systemic androgen blockade. However, response rates withlong-term (10 years) therapy are only around 50%. Overall, despiteconsiderable research in this area, there is still no adequate therapyfor hair loss.

In addition, unwanted hair growth is a cosmetic issue many people dealwith on a regular basis. Unwanted hair growth on the face, legs, arms,chest or back is a growing cosmetic problem. Many people use lasertherapy, waxing or other therapies to remove unwanted hair. There arecurrently no topical pharmaceuticals to limit hair growth.

Collagenopathies represent a large number of diseases in which collagenstructure or formation is not normal. This group of diseases results ina broad spectrum of symptoms including bone defects, vascular defects,and skin defects. Many of these diseases have no or only ineffectivetreatments available.

For example, osteogenesis imperfecta (OI), also known as brittle bonedisease, is caused by an inborn mutation of type I collagen.Approximately 25,000 to 50,000 Americans are affected and the effects ofthe disease range from mild, in which many individuals are unaware ofthe disease, to severe in which individuals cannot live a normal lifedue to recurrent broken bones. Most OI patients carry a mutation whichcauses an amino acid change in collagen changing a glycine to a bulkieramino acid which results in disruption of the triple helix structure ofthe collagen and under-twisting. The body may respond by hydrolyzing thecollagen and this may result in a reduction in bone strength. There iscurrently no cure and few treatments for OI.

SUMMARY

Provided herein are methods of delivering therapeutic agents byadministering compositions including a bacterial collagen-bindingpolypeptide segment linked to the therapeutic agent to subjects in needof treatment with the therapeutic agent. In these methods, thetherapeutic agent is not a PTH/PTHrP receptor agonist or antagonist,basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF)and the bacterial collagen-binding polypeptide segment delivers theagent to sites of partially untwisted or under-twisted collagen.

In another aspect, methods of treating a subject with a collagenopathy,such as osteogenesis imperfecta, by administering a compositioncomprising a bacterial collagen-binding polypeptide segment linked to aPTH/PTHrP receptor agonist to a subject in an amount effective to treatthe collagenopathy are provided. The bacterial collagen-bindingpolypeptide segment delivers the agent to sites of partially untwistedor under-twisted collagen.

In yet another aspect, methods of treating hyperparathyroidism byadministering a composition comprising a bacterial collagen-bindingpolypeptide segment linked to a PTH/PTHrP receptor agonist to a subjectare provided.

In still a further aspect, methods of slowing hair growth or regrowthafter removal by administering a composition comprising a bacterialcollagen-binding polypeptide segment linked to a PTH/PTHrP receptorantagonist to a subject are provided.

In a still further aspect, methods of increasing hair growth or thespeed of hair re-growth after removal or loss by administering acomposition comprising a bacterial collagen-binding polypeptide segmentlinked to a PTH/PTHrP receptor agonist to a subject are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the office upon request and paymentof the necessary fee.

FIG. 1 is a sequence alignment showing the alignment of several M9Bbacterial collagenases from the Bacillus and Clostridium families. Theresidues shown in blue are important for collagen binding activity,those shown in green are important for maintaining the architecture orprotein folding. Both of these are also underlined for the top andbottom sequences. Residues shown in red are critical for Ca²⁺ bindingand those in orange are critical for positioning the Ca²⁺ bindingresidues. The sequences are also included in the Sequence Listing filedherewith as SEQ ID NOs: 13-34, where SEQ ID NO: 13 is the first listedColG s3b sequence and SEQ ID NO: 34 is the ColH s3 sequence.

FIG. 2 is a set of drawings showing the chemical structures ofsynthesized peptides.

FIG. 3A is a graph showing the circular dichroism spectra of thecollagenous peptides measured at 4° C.

FIG. 3B is a graph showing the thermal denaturation profile of thevarious collagenous peptides. The temperature was increased at the rateof 0.3° C./min.

FIG. 4A is a graph showing the scattering profile with the intensityI(Q) plotted against the scattering vector Q.

FIG. 4B is a graph showing the pair-distance distribution function P(r)in the real space obtained using GNOM for [PROXYL-(POG)₃POA(POG)₆]₃:CBDcomplex (Red), [PROXYL-(POG)₄POA(POG)₅]₃:CBD complex (Blue),[PROXYL-(POG)₅POA(POG)₄]₃:CBD complex (Green),[PROXYL-(POG)₆POA(POG)₃]₃:CBD complex (Orange) and[11PROXYL-(POG)₃PCG(POG)₄]₃:CBD complex (Cyan).

FIG. 5 is a set of plots showing HSQC NMR data obtained using thecollagen binding domain (CBD)—collagenous peptide interactions. FIG. 5Ashows an overlay of ¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQCspectrum of [(POG)₁₀]₃:CBD complex (green) at 1:1 ratio. Amide resonanceof V973, G975 and S979 are present during this titration. FIG. 5B showsan overlay of ¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQCspectrum of [PROXYL-(POG)₆POA(POG)₃]₃:CBD complex (red) at 1:1 ratio.Amide resonances of V973, G975 and S979 disappeared because of theirproximity to the spin-labeled group. FIG. 5C is a cartoon showing thestructure of CBD and the CBD residues that are line broadened upontitration with [PROXYL-(POG)₆POA(POG)₃]₃.

FIG. 6 is a set of plots showing HSQC NMR data obtained using theCBD—collagenous peptide interactions. FIG. 6A shows an overlay of ¹H-¹⁵NHSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of [(POG)₁₀]₃:CBDcomplex (green) at 1:1 ratio. Amide resonances of Q972, V973, G975 andS979 are present during this titration. FIG. 6B shows an overlay of¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of[PROXYL-(POG)₅POA(POG)₄]₃:CBD complex (red) at ratio 1:1. Amideresonances of Q972, V973, G975 and S979 are line broadened due to thePROXYL moiety. FIG. 6C is a cartoon of the structure of CBD showing theCBD residues that are uniquely line broadened upon titration with[PROXYL-(POG)₅POA(POG)₄]₃. FIG. 6D shows an overlay of ¹H-¹⁵N HSQCspectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of [(POG)₁₀]₃:CBDcomplex (green) at 1:1 ratio. Amide resonances of L946, Q972, V973, G975and S979 are present during this titration. FIG. 6E shows an overlay of¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of[PROXYL-(POG)₄POA(POG)₅]₃:CBD complex (red) at 1:1 ratio. Amideresonances of L946, Q972, V973, G975 and S979 disappeared because of thespin-label. FIG. 6F shows an overlay of ¹H-¹⁵N HSQC spectrum of CBD(black) and ¹H-¹⁵N HSQC spectrum of [(POG)₄POA(POG)₅]₃:CBD (cyan) atratio 1:1. In the absence of spin label, amide resonances of L946, Q972,V973, G975 and S979 are not line broadened. FIG. 6G shows an overlay ofH-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of[(POG)₁₀]₃:CBD complex (green) at 1:1 ratio. Amide resonances of L946,G953, Q972, V973, D974, G975, N976, V978, S979 are present during thistitration. FIG. 6H shows an overlay of ¹H-¹⁵N HSQC spectrum of CBD(black) and ¹H-¹⁵N HSQC spectrum of [PROXYL-(POG)₃POA(POG)₆]₃:CBDcomplex (red) at ratio 1:1. Amide resonances of L946, G953, Q972, V973,D974, G975, N976, V978, S979 are line broadened due to the PROXYLmoiety. FIG. 6I is a cartoon of the structure of CBD showing the CBDresidues that are line broadened by the spin label of[PROXYL-(POG)₃POA(POG)₆]₃.

FIG. 7 is a set of graphs showing the intensity drop of (FIG. 7A) Q972,(FIG. 7B) G975, (FIG. 7C) S979 and (FIG. 7D) L924 on CBD as a functionof increasing concentrations of mini-collagen i.e. [(POG)₁₀]₃ (black),[PROXYL-(POG)₆POA(POG)₃]₃ (red), [PROXYL-(POG)₅POA(POG)₄]₃ (blue),[PROXYL-(POG)₄POA(POG)₅]₃ (green), and [PROXYL-(POG)₃POA(POG)₆]₃ (cyan).

FIG. 8 is a set of plots showing HSQC NMR data obtained using theCBD—collagenous peptide interactions. FIG. 8A shows an overlay of ¹H-¹⁵NHSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of [(POG)₁₀]₃:CBDcomplex (green) at 1:1 ratio. Amide resonances of S906, S997 and G998are present during this titration. FIG. 8B shows an overlay of ¹H-¹⁵NHSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of[(POG)₄POA(POG)₅C-PROXYL]₃:CBD complex (red) at ratio 1:1. Amideresonances of S906, S997 and G998 are line broadened due to the PROXYLmoiety. FIG. 8C shows an overlay of ¹H-¹⁵N HSQC spectrum of CBD (black)and ¹H-¹⁵N HSQC spectrum of [(POG)₄POA(POG)₅C-carbamidomethyl]₃:CBD(cyan) at 1:1 ratio. In the absence of spin label, amide resonances ofS906, S997 and G998 are not line broadened. FIG. 8D is a cartoon of thestructure of CBD showing the CBD residues that are line broadened due tothe spin label of [(POG)₄POA(POG)₅C-PROXYL]₃. Amide resonances of S906,S997 and G998 (red) disappeared upon titration with[(POG)₄POA(POG)₅-PROXYL]₃. FIG. 8E shows an overlay of ¹H-¹⁵N HSQCspectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of [(POG)₁₀]₃:CBDcomplex (green) at 1:1 ratio. Amide resonances of S906, Q972, V973,G975, S979, S997 and G998 are present during this titration. FIG. 8Fshows an overlay of ¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQCspectrum of [11PROXYL-(POG)₃PCG(POG)₄]₃:CBD complex (red) at 1:1 ratio.Amide resonances of S906, Q972, V973, G975, S979, S997 and G998disappeared because of the spin-label. FIG. 8G shows an overlay of¹H-¹⁵N HSQC spectrum of CBD (black) and ¹H-¹⁵N HSQC spectrum of[(POG)₃PCG(POG)₄]₃:CBD (cyan) at ratio of 1:1. Resonances of S906, Q972,V973, G975, S979, S997 and G998 are intact in the absence of the spinlabel. FIG. 8H is a cartoon of the structure of CBD showing the residuesthat are line broadened upon titration with [11PROXYL-(POG)₃PCG(POG)₄]₃.Only amide resonances of S906, R929, S997, and G998 (red) disappeared at0.2:1 ratio. When the peptide ratio was raised to 0.3:1, additionalresonances of V973, G975, S979 (blue) disappeared.

FIG. 9 is a set of structure drawings derived from SAXS scatteringprofiles using ab initio simulated annealing calculations for (FIG. 9A)[PROXYL-(POG)₃POA(POG)₆]₃:CBD complex, (FIG. 9B)[PROXYL-(POG)₄POA(POG)₅]₃:CBD complex, (FIG. 9C)[PROXYL-(POG)₅POA(POG)₄]₃:CBD complex and (FIG. 9D)[PROXYL-(POG)₆POA(POG)₃]₃:CBD complex, (FIG. 9E)[(POG)₄POA(POG)₅C-PROXYL]₃:CBD complex, (FIG. 9F)[(POG)₄POA(POG)₅C-carbamidomethyl]₃:CBD. The Gly→Ala mutation sites arehighlighted. FIG. 9G and FIG. 9H show two probable binding modes of[11PROXYL-(POG)₃PCG(POG)₄]₃: CBD complex.

FIG. 10 is a set of plots showing HSQC NMR data obtained using theCBD-collagenous peptide interactions. FIG. 10A is an overlay of ¹H-¹⁵NHSQC spectrum of [POGPO-¹⁵N-G-(POG)₈]₃ (black) with ¹H-¹⁵N HSQC spectrumof [POGPO-¹⁵N-G-(POG)₈]₃:CBD complex (red) at 1:1 ratio. FIG. 10B showsan overlay of ¹H-¹⁵N HSQC spectrum of [POGPO-¹⁵N-G-(POG)₂₋POA-(POG)₅]₃(black) with ¹H-¹⁵N HSQC spectrum of[POGPO-¹⁵N-G-(POG)₂₋POA-(POG)₅]₃:CBD complex (red) at 1:1 ratio. FIG.10C shows an overlay of ¹H-¹⁵N HSQC spectrum of [(POG)₈-PO-¹⁵N-G-POG]₃(black) with ¹H-¹⁵N HSQC spectrum of [(POG)₈-PO-¹⁵N-G-POG]₃:CBD complex(red) at 1:1 ratio. FIG. 10D shows an overlay of ¹H-¹⁵N HSQC spectrum of[(POG)₄-POA-PO-¹⁵N-G-POG]₃ (black) with ¹H-¹⁵N HSQC spectrum of[(POG)₄-POA-PO-¹⁵N-G-POG]₃:CBD complex (red) at 1:1 ratio.

FIG. 11 shows the tissue distribution of S³⁵-PTH-CBD 1 hour and 12 hoursafter subcutaneous injection. Note the skin outline.

FIG. 12 is a set of photographs documenting the hair growth on the backof mice at day 36 after depilation, treatment groups as indicated. FIG.12A is control; FIG. 12B is CYP treatment alone; FIG. 12C is CYP and PTHantagonist; FIG. 12D is CYP and PTH agonist (Antagonist=PTH(7-33)-CBD,Agonist=PTH-CBD).

FIG. 13 is a set of photographs showing the histology at Day 36 afterthe indicated treatment. Skin samples were taken from the dorsal regionand processed for Hematoxylin and Eosin (H&E) staining. Representativesections are shown from each treatment group as indicated. FIG. 13A iscontrol; FIG. 13B is CYP alone; FIG. 13C is CYP and PTH agonist; FIG.13D is CYP and PTH antagonist (Antagonist=PTH(7-33)-CBD,Agonist=PTH-CBD).

FIG. 14 is a graph showing the hair follicle counts per high poweredfield. Anagen VI hair follicles were counted by two independentobservers in a blinded fashion. Results are expressed as mean+/−standarddeviation. **=p<0.01 vs. no chemo ANOVA followed by Dunnett's test.(Antagonist=PTH(7-33)-CBD, Agonist=PTH-CBD).

FIG. 15 is a set of photographs showing the hair growth on the back ofthe mice after each of the indicated treatments (FIG. 15A ischemotherapy alone; FIG. 15B is no chemotherapy control; FIG. 15C ischemotherapy and 100 mcg/kg PTH agonist-CBD; FIG. 15D is chemotherapyand 320 mcg/kg PTH agonist-CBD; FIG. 15E is chemotherapy and 1000 mcg/kgPTH agonist-CBD) and a graph (FIG. 15F) showing the results of a greyscale analysis of the hair at the injection site over time after theinjection.

FIG. 16 is a set of photographs showing the hair on the back of miceafter the indicated treatment without prior depilation. FIG. 16A showschemotherapy alone; FIG. 16B shows chemotherapy and PTH-CBD and FIG. 16Cshows no chemotherapy control.

FIG. 17 is a set of photographs and a graph (FIG. 17A) showing the greyscale analysis of hair growth on the backs of mice comparing theindicated treatments with the PTH-CBD being administered prior to thechemotherapy as opposed to after chemotherapy began. FIG. 17B showschemotherapy alone; FIG. 17C shows PTH-CBD prophylaxis and FIG. 17Dshows chemotherapy and PTH-CBD therapy.

FIG. 18 is a photograph of three mice 13 days after waxing to removehair and treatment with PTH-CBD, PTH antagonist-CBD or vehicle alone.

FIG. 19 is a set of photographs of mice showing hair regrowth in a modelof alopecia areata after treatment with a control or with PTH-CBD.

FIG. 20 is a graph showing the endogenous parathyroid hormone levels inovarectomized aged rats injected with a single dose of human PTH-CBD 6months prior to sacrifice.

DETAILED DESCRIPTION

Methods of delivering a therapeutic agent by administering a compositioncomprising a bacterial collagen-binding polypeptide segment linked to atherapeutic agent to a subject in need of treatment with the therapeuticagent are provided herein. In this embodiment, the therapeutic agent isnot a PTH/PTHrP receptor agonist or antagonist and is not a bFGF or EGFpolypeptide. The bacterial collagen-binding polypeptide segment deliversthe therapeutic agent to sites of partially untwisted or under-twistedcollagen.

In addition, methods of treating collagenopathies, such as osteogenesisimperfecta (OI), by administering a composition comprising a bacterialcollagen-binding polypeptide segment linked to a PTH/PTHrP receptoragonist to a subject in need of treatment for a collagenopathy areprovided. Collagenopathies include but are not limited to osteogenesisimperfecta, Stickler's syndrome, Ehlers-Danlos syndrome, Alport'ssyndrome, Caffey's disease, and localized collagen or cartilage damage.Many of these diseases are caused by genetic defects that result in thecollagen in certain tissues being under twisted or partially untwisted.

For example, individuals with OI carry a mutation which causes an aminoacid change in collagen changing a glycine to a bulkier amino acid whichresults in disruption of the triple helix structure of the collagen andunder-twisting of the collagen. In the Examples, we demonstrate that thebacterial collagen-binding polypeptides described herein target and bindto these areas of under-twisted collagen. Thus, use of thecollagen-binding polypeptides described herein to deliver a therapeuticagent capable of treating OI to the sites of under-twisted collagen mayallow more effective treatment.

The collagen-binding polypeptide segment and the therapeutic agent maybe chemically cross-linked to each other or may be polypeptide portionsof a fusion protein. The terms “fusion protein” and “fusion polypeptide”may be used to refer to a single polypeptide comprising two functionalsegments, e.g., a collagen-binding polypeptide segment and a polypeptidebased therapeutic agent, such as PTH/PTHrP receptor agonist polypeptidesegment. The fusion proteins may be any size, and the single polypeptideof the fusion protein may exist in a multimeric form in its functionalstate, e.g., by cysteine disulfide connection of two monomers of thesingle polypeptide. A polypeptide segment may be a synthetic polypeptideor a naturally occurring polypeptide. Such polypeptides may be a portionof a polypeptide or may comprise one or more mutations. The twopolypeptide segments of the fusion proteins can be linked directly orindirectly. For instance, the two segments may be linked directlythrough, e.g., a peptide bond or chemical cross-linking, or indirectly,through, e.g., a linker segment or linker polypeptide. The peptidelinker may be any length and may include traditional or non-traditionalamino acids. For example, the peptide linker may be 1-100 amino acidslong, suitably it is 5, 10, 15, 20, 25 or more amino acids long suchthat the collagen binding portion of the fusion polypeptide can mediatecollagen binding and the therapeutic agent can have its therapeuticeffect. Peptide linkers may include but are not limited to a PKD(polycystic kidney disease) domain from a collagenase or other proteinsuch as in SEQ ID NO: 2, a GST or His-tag, or a Ser or Gly linker.

The collagen-binding polypeptide segment is a polypeptide that bindscollagen and may be part of a larger fusion protein, bioactive agent, orpharmaceutical agent. Determination of whether a composition,polypeptide segment, fusion protein, or pharmaceutical or bioactiveagent binds collagen can be made as described in U.S. Patent PublicationNo. 2010/0129341, which is incorporated herein by reference in itsentirety. Briefly, it is incubated with collagen in binding buffer, andthe mixture is then filtered through a filter that would otherwise allowit to pass through but that blocks the collagen and therefore holds backmaterials that bind to the collagen. The filtrate is then assayed forthe presence of the composition, polypeptide segment, fusion protein, orpharmaceutical or bioactive agent. Suitably, at least 80%, 85%, 90%,95%, 98% or more suitably at least 99% of the collagen-bindingcomposition, polypeptide segment, fusion protein, or pharmaceutical orbioactive agent is retained by the filter in this assay, as compared towhen the filtration is performed without collagen.

The collagen-binding polypeptide segment may be a bacterialcollagen-binding polypeptide segment. It may be a Clostridiumcollagen-binding polypeptide segment. The collagen-binding polypeptidesegment may be a segment of a collagenase, or a bacterial collagenase,or a Clostridium collagenase. Suitably the polypeptide segment is only aportion of the collagenase and the collagen-binding polypeptide segmentdoes not have collagenase activity. The collagen-binding polypeptide maybe a bacterial M9B (including those derived from Bacillus spp. andClostridium spp.) or M9A (including those derived from Vibrio spp.)collagen-binding protein or a collagen-binding peptide derived from sucha protein. By “derived from” we mean that the peptide is a fragment ofthe full-length protein, a peptide that has amino acid changes relativeto the wild-type protein or a combination thereof. The key is that thepeptide retains the ability to bind collagen. For example, a peptide maybe derived from a protein by selecting a region of the protein capableof binding to collagen. Compositions including a bacterial collagenaseas a collagen binding peptide are described in US Patent Publication No.2010/0129341, which is hereby incorporated herein by reference in itsentirety.

FIG. 1 shows a sequence alignment of the collagen-binding region ofseveral M9B bacterial collagen-binding proteins included as SEQ ID NOs:13-34. As can be seen from the sequence alignment, these proteins have arelatively small amount of sequence identity (about 30%), but they allbind to collagen in a similar fashion and are believed to have similarconformation as discussed in the Examples. Thus any of the peptidesshown in FIG. 1 or collagen-binding fragments thereof can be used in thecompositions and methods described herein. In FIG. 1, the amino acidresidues critical for the conformation of the peptide and for thecollagen-binding activity are underlined and shown in green and bluerespectively. The key amino acid residues for collagen-binding are atyrosine or phenylalanine at position 970 of ColG, position 977 of theColH sequence of SEQ ID NO: 1 (position 937 in FIG. 1) or a similarposition of one of the sequences shown in FIG. 1; a tyrosine at position994 of ColG, position 1000 of the ColH sequence of SEQ ID NO: 1(position 962 in FIG. 1) or a similar position of one of the sequencesshown in FIG. 1; a tyrosine, phenylalanine or histidine at position 996of ColG, position 1002 of the ColH sequence of SEQ ID NO: 1 (position964 in FIG. 1) or a similar position of one of the sequences shown inFIG. 1. Thus a peptide with relatively low sequence identity, sharingthe structure and function of the ColG protein may also be used as acollagen binding domain (CBD) herein.

In one embodiment, the collagenase is ColH, SEQ ID NO: 6. Thecollagen-binding polypeptide segment may be or may include residues901-1021 of SEQ ID NO:6 (residues 34-158 of SEQ ID NO:1), or a fragmentof residues 34-158 of SEQ ID NO:1 at least 8, 10, 12, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acid residues in length.The collagen-binding polypeptide segment is at least 50%, 60%, 70%, 80%,or at least 85%, at least 90%, at least 95%, at least 96%, at least 98%,or at least 99% identical to residues 34-158 of SEQ ID NO: 1. Thecollagen-binding polypeptide segment may be or may include residues807-1021 of SEQ ID NO:6 (residues 37-251 of SEQ ID NO:2), or a fragmentof residues 807-1021 of SEQ ID NO:6 at least 8, 10, 12, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acid residues in length.Residues 807-901 comprise the polycystic kidney disease (PKD) domain ofthe collagen-binding protein. Those of skill in the art will appreciatethat other linkers could be used to link the collagen-binding peptide toa therapeutic agent as outlined above. The collagen-binding polypeptidesegment may be or may comprise a fragment of residues 901-1021 of SEQ IDNO:6, e.g., a fragment of at least 8, at least 10, at least 20, at least30 at least 40, or at least 50 consecutive amino acid residues ofresidues 901-1021 of SEQ ID NO:6. Suitably the collagen-bindingpolypeptide consists of residues 894-1008, 894-1021, 901-1021, or901-1008 of SEQ ID NO: 6 or a homolog thereof as shown by the sequencealignment in FIG. 9.

Among other proteins the collagen-binding segment can be derived fromare ColG (Matsushita et al., (1999) J. Bacteriol. 181:923-933), a classI collagenase from Clostridium histolyticum. ColH is a class IIcollagenase (Yoshihara et al., (1994) J. Bacteriol. 176: 6489-6496). Thecollagen-binding polypeptide segment may also be a polypeptide segmentfrom any one of the protein sequences provided in FIG. 1 which alignscollagen-binding peptides from members of Clostridium and Bacillus.Those of skill in the art will appreciate that other members of thiscollagen-binding protein family may be useful in the methods describedherein.

The therapeutic agents linked to the collagen-binding polypeptide may beany suitable pharmaceutical or other active agent, including but notlimited to, osteogenic promoters, antimicrobials, anti-inflammatoryagents, polypeptides such as recombinant proteins, cytokines orantibodies, small molecule chemicals or any combination thereof.Suitably the therapeutic agents are capable of promoting bone growth,decreasing inflammation, promoting collagen stability. Suitably, thetherapeutic agent is one whose therapeutic effect is in the region ofcollagen or damaged collagen. The therapeutic agent may include, but isnot limited to, bone morphogenic protein (BMP), G-CSF, FGF, BMP-2,BMP-3, FGF-2, FGF-4, anti-sclerostin antibody, growth hormone, IGF-1,VEGF, TGF-β, KGF, FGF-10, TGF-α, TGF-β1, TGF-β receptor, CT, GH, GM-CSF,EGF, PDGF, celiprolol, activins and connective tissue growth factors. Inalternative embodiments, the active agent may be a PTH/PTHrP receptoragonist or antagonist.

Bone loss due to a collagenopathy such as osteogenesis imperfecta,Stickler's syndrome or others which put an individual at higher risk fora bone fracture due to a collagen defect could be treated byadministration of a bone anabolic peptide. The CBD may target the boneanabolic agents to sites where the collagen is malformed and thus mayprevent fracture.

Vascular fragility due to defects such as Ehlers-Danlos syndrome typeIV, Alport's syndrome or other diseases where blood vessel rupture ismore likely due to a defect in collagen formation may be administeredpeptides that stimulate vascular growth or repair. The CBD will targetthe peptide to the areas having collagen damage and these areas arelikely to have damaged vessels. The therapeutic agents will stimulategrowth and repair at the site of damage and prevent vessel rupture.

Skin fragility due to disorders such as Ehlers-Danlos syndrome, Caffey'sdisease or other diseases where weakening of the skin due to a collagendefect leads to hyperelasticity, easy bruising or poor wound healing.Dermal and epidermal growth factors may serve as therapeutic agentswhich when linked to CBD and delivered to areas of damaged collagen willstimulate growth and repair of the skin, preventin striae and improvinghealing.

Collagen defects may also lead to cartilage malformation orinsufficiency. Cartilage growth factors could be delivered locally tosites of damaged cartilage to aid in repair and restore function.

The PTH/PTHrP receptor agonist polypeptide segment may be a syntheticpolypeptide or a naturally occurring polypeptide. Such polypeptides maybe a portion of a polypeptide or may comprise one or more mutations. Themutations may make the PTH/PTHrP receptor agonist a better or worseagonist as compared to the wild-type PTH/PTHrP. Agonist activity withthe PTH/PTHrP receptor can be assayed as described in Example 3 below bya cAMP stimulation assay. An agonist will stimulate cAMP synthesis inthe assay described. Suitably, an agonist can activate receptor activityat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even 110%or 120% as much as wild-type PTH(1-34).

The PTH/PTHrP receptor agonist polypeptide segment is a PTH or PTHrPpolypeptide segment. One human isoform of PTH is SEQ ID NO:7. One humanisoform of PTHrP is SEQ ID NO:8. While the human isoforms are provided,those of skill in the art will appreciate that other non-human-derivedisoforms may be used as well. Such non-human-derived isoforms may beable to interact with human PTH/PTHrP receptor and vice versa. ThePTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-33 of SEQ ID NO: 1 (residues 1-33 of PTH (SEQ ID NO:7)). ThePTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-34 of PTH (SEQ ID NO:7). In other embodiments, it is afragment of residues 1-34 of PTH (SEQ ID NO:7). In other embodiments,the PTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-84 of PTH (SEQ ID NO:7). In other embodiments, the PTH/PTHrPreceptor agonist polypeptide segment may be or may include residues 1-14of PTH (SEQ ID NO:7). In still other embodiments, the PTH/PTHrP receptoragonist is a PTH or PTHrP polypeptide segment for any other species.

The PTH/PTHrP receptor antagonist can include in one embodimentPTH(7-34), i.e., residues 7-34 of PTH (SEQ ID NO:7). In anotherembodiment, it is or includes residues 7-33 of PTH (SEQ ID NO:7). Inother embodiments, it is a fragment of residues 7-34 of SEQ ID NO: 8. Inanother embodiment, the PTH/PTHrP receptor antagonist includesPTH(7-14), i.e., residues 7-14 of PTH (SEQ ID NO:7). In anotherembodiment, the PTH/PTHrP receptor antagonists include ((−1)-33) ofPTH/PTHrP. In another embodiment, the PTH/PTHrP receptor antagonistsinclude residues 1-14 of PTH with an N-terminal extension. Adding anN-terminal extension to PTH or active N-terminal fragments of PTHconverts the PTH peptides to antagonists. The N-terminal extension canbe 1, 2, 3, 4, 5, or more amino acids in length. The identity of theamino acids in the N-terminal extension is typically not important. Inone embodiment, the PTH/PTHrP receptor antagonist includes residues 1-33of PTH with a Gly-Ser extension at the N-terminus (SEQ ID NO: 11). Inanother embodiment, the PTH/PTHrP receptor antagonist includesPTHrP(7-34), i.e., residues 7-34 of SEQ ID NO:8, or a fragment ofresidues 7-34 of SEQ ID NO:8. In another embodiment, the PTH/PTHrPreceptor antagonist includes mouse TIP(7-39) (See Hoare S R, Usdin T B.2002. Specificity and stability of a new PTH1 receptor antagonist, mouseTIP(7-39). Peptides 23:989-98.). Other PTH/PTHrP receptor antagoniststhat may be used in the fusion proteins are also disclosed in Hoare etal. The PTH/PTHrP receptor antagonist may be a fragment of at least 8,10, 12 or more amino acids from residues 1-34 of SEQ ID NO:7. In otherembodiments the PTH/PTHrP receptor antagonist may be PTH/PTHrP receptorantagonist polypeptide from another species.

In one embodiment, the therapeutic agent or PTH/PTHrP receptor agonistor antagonist polypeptide segment is N terminal to the collagen-bindingpolypeptide segment in the fusion protein. That is, the two polypeptidesegments each have an N-terminal and a C-terminal, and the N-terminal ofthe collagen-binding polypeptide segment is linked directly orindirectly, e.g., through a linker polypeptide segment (such as PKD, aGlycine or Serine linker) to the C-terminal of the therapeutic agent orPTH/PTHrP agonist or antagonist polypeptide segment.

The fusion proteins described above comprising (a) a collagen-bindingpolypeptide segment linked to (b) a therapeutic agent or a PTH/PTHrPreceptor agonist or antagonist polypeptide segment can be replaced bypharmaceutical agents comprising (a) a collagen-binding polypeptidesegment linked to (b) a therapeutic agent or PTH/PTHrP receptor agonistor a non-peptidyl PTH/PTHrP receptor agonist. An example of anon-peptidyl PTH/PTHrP receptor agonist is compound AH3960 (Rickard etal., (2007) Bone 39:1361-1372).

AH3960 contains two amino groups. Amino groups in small chemicalmolecules such as AH3960 can be used to cross-link the therapeutic agentto amino groups on the collagen-binding polypeptide segment through across-linker such as DSG (disuccinimidyl glutarate) or through thecombination of SANH (succinimidyl-4-hydrazinonicotinate acetonehydrazone) and SFB (succinimidyl-4-formyl benzoate). Therapeutic agentscan be cross-linked through their amino group to a carboxyl group of thecollagen-binding polypeptide segment by EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) or viceversa. These cross-linking products are available from Pierce(piercenet.com, Thermo Fisher Scientific Inc., Rockford, Ill.).Protocols and reaction conditions are also available in the productliterature from Pierce (piercenet.com).

In another embodiment of the pharmaceutical agents comprising (a) acollagen-binding polypeptide segment; linked to (b) a polypeptidetherapeutic agent or a PTH/PTHrP receptor agonist or antagonistpolypeptide segment, segment (a) and segment (b) are separatepolypeptides, and the two polypeptides are linked by chemicalcross-linking. The two polypeptides can be cross-linked through aminogroups by reagents including DSG (disuccinimidyl glutarate) orglutaraldehyde. They can also be cross-linked through amino groups byderivatizing one polypeptide with SANH(succinimidyl-4-hydrazinonicotinate acetone hydrazone) and the otherwith SFB (succinimidyl-4-formyl benzoate), and then mixing the twoderivatized polypeptides to cross-link. The two polypeptides can becross-linked between an amino group of one polypeptide and a carboxyl ofthe other by reaction with EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Thepolypeptides can also be cross-linked (e.g., covalently coupled) by anyother suitable method known to a person of ordinary skill in the art.These cross-linking reagents are available from Pierce (piercenet.com,Thermo Fisher Scientific Inc., Rockford, Ill.). Protocols and reactionconditions are also available in the product literature from Pierce(piercenet.com). These and other applicable cross-linking methods aredescribed in U.S. published patent applications 2006/0258569 and2007/0224119.

Also provided herein are methods of treating hyperparathyroidism byadministering PTH-CBD to a subject in need of treatment forhyperparathyroidism. In one embodiment the PTH administered to thesubject may be a PTH from a different species. As shown in the Examplesa single administration of CBD-PTH to ovarectomized aged rats was ableto reduce the amount of endogenous PTH produced by the animal. Thus,administration of PTH-CBD to individuals suffering fromhyperparathyroidism may experience a decrease in symptoms associatedwith hyperparathyroidism and have decreased levels of PTH afteradministration of PTH-CBD.

The effects of PTH agonists and antagonists on hair growth have beenstudied for over almost 15 years. PTH has a common receptor withPTH-related peptide (PTHrP), which is normally produced by dermalfibroblasts. PTHrP affects keratinocyte proliferation/differentiationand modulates the hair cycle. Most of the testing on hair growth effectshas been performed with PTH antagonists, as indications from initialtesting were that these were the most effective agents. Both injectedand topical formulations have been tested in animal models ofchemotherapy-induced alopecia and in the SKH-1 hairless mouse. Part ofthe effect of PTH antagonists on hair growth is to transition the hairfollicles into a dystrophic catagen stage, which protects them fromchemotherapeutic damage. However, clinical trials of topical PTHantagonists for chemotherapy-induced alopecia by IGI Pharmaceuticalswere discontinued in phase 2 because of limited efficacy. Thus newcompositions for treating alopecia are needed.

The problems of delivery and retention of PTH to the skin can beovercome by using collagen-targeted PTH analogs. To accomplish this, wesynthesized several fusion proteins of different PTH agonists andantagonists linked to a collagen binding domain derived from the ColH1collagenase of Clostridium histolyticum. In the studies described in theExamples, we found that the agonist compound PTH-CBD promotes transitionof hair follicles to the anagen phase and has potent effects on hairgrowth. The antagonist compound PTH(7-33)-CBD had little effect on hairgrowth in chemotherapy models and had a deleterious effect on hairregrowth after depilation. Compounds such as PTH-CBD, which promoteanagen phase transition of hair follicles, have been sought after due totheir potential to treat a large variety of disorders of hair loss.PTH-CBD appears to have a similar mechanism of action to cyclosporine,which also promotes transition of hair follicles to anagen phase,although the mechanism is less likely to be the result of direct effectson WNT signaling. While clinical use of cyclosporine for this purpose islimited by systemic toxicity, PTH-CBD has not shown toxic effects, evenwith systemic administration.

Thus in another aspect, methods of increasing hair growth are providedherein. The methods include administering a CBD linked to a PTH/PTHrPreceptor agonist to a subject in need of treatment to induce hair growthor stop hair loss. The method is applicable to individuals withalopecia, including chemotherapy induced alopecia, but also alopeciaareata, alopecia caused by male pattern baldness, polycystic ovariansyndrome or other hair loss. The compositions may be administeredlocally or topically to treat hair loss.

In another aspect, methods of slowing hair growth or regrowth after ahair removal procedure by administering a CBD linked to a PTH/PTHrPreceptor antagonist to a subject are provided. In one embodiment, thePTH antagonist composition is applied locally, topically. The PTHantagonist may be applied after a hair removal procedure to prevent orslow hair regrowth. As described in the Examples, we have demonstratedthat hair regrowth is slowed after waxing in animals treated withCBD-PTH antagonist as compared to control animals treated with PTH-CBDor vehicle alone. The compositions may be administered locally ortopically to block hair growth.

The compositions described herein may be administered by any means knownto those skilled in the art, including, but not limited to, oral,topical, intranasal, intraperitoneal, parenteral, intravenous,intramuscular, intradermal or subcutaneous. Thus the compositions may beformulated as an ingestible, injectable, topical or suppositoryformulation. The composition may be formulated for administration byinjection to result in systemic administration or local administration.The compositions may also be delivered with in a liposomal ortime-release vehicle. The compositions may also be delivered in asite-directed delivery vehicle, such as but not limited to, a targetedliposome or an absorbable collagen sponge carrier or other implant.

The inventors have found that when administering compositions includinga CBD subcutaneously it binds locally at the site of injection if thecomposition is dissolved in neutral pH buffer. But if the composition isdissolved in a low pH buffer, for example a buffer having pH 5.0 or pH4.5 or below, the collagen-binding domain does not bind collagen, andthe composition has time to disperse systemically before it bindscollagen elsewhere in the body at neutral pH. Thus systemicadministration of the compositions involves administering thecomposition dissolved in buffer or aqueous solution at a pH lower thanabout 5.0 or at pH 4.5 or below. In another embodiment, systemicadministration of the compositions involves administering the fusionproteins dissolved in aqueous solution at pH lower than about 6.0.Alternatively, if the skin condition is localized, the compositionsdescribed herein may be administered in a buffer with a pH of 6.0, 6.5,7.0, 7.5 or above in order to allow for localized delivery of thecompositions to the affected area of the skin.

Pharmaceutical compositions for topical administration may also beformulated using methods and compositions such as those available tothose skilled in the art. For example, gels, creams or liposomepreparations may be suitable for topical delivery. These deliveryvehicles may be formulated to mediate delivery to the lower layers ofthe skin or to allow for extended release of the pharmaceutical at thesite of application.

The compositions can be administered as a single dose or as divideddoses. For example, the composition may be administered two or moretimes separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days,three days, four days, one week, two weeks, or by three or more weeks.Optionally, such treatment may be repeated, for example, every 1, 2, 3,4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The composition is expected tobe more effective than a comparable or control composition comprisingthe therapeutic agent or a PTH/PTHrP receptor agonist that is not linkedto a collagen-binding protein. In one embodiment, a smaller amount ofthe composition may be used or the composition may be administered lessfrequently than a comparable composition comprising the therapeuticagent or a PTH/PTHrP receptor agonist which is not linked to acollagen-binding protein.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The individual doses of pharmaceutical agents comprising acollagen-binding polypeptide segment linked to a therapeutic agent maybe approximately the same on a molar basis as doses used for thetherapeutic agent alone. It is expected that the pharmaceutical agentscomprising a collagen-binding polypeptide segment linked to atherapeutic agent may be administered less frequently, because linkingthe agent to the collagen-binding polypeptide segment gives it much moreprolonged activity in vivo.

Administration of the compositions to a subject in accordance with theinvention appears to exhibit beneficial effects in a dose-dependentmanner. Thus, within broad limits, administration of larger quantitiesof the compositions is expected to achieve increased beneficialbiological effects than administration of a smaller amount. Moreover,efficacy is also contemplated at dosages below the level at whichtoxicity is seen.

It will be appreciated that the specific dosage administered in anygiven case will be adjusted in accordance with the compositions beingadministered, the disease to be treated or inhibited, the condition ofthe subject, and other relevant medical factors that may modify theactivity of the agent or the response of the subject, as is well knownby those skilled in the art. For example, the specific dose for aparticular subject depends on age, body weight, general state of health,diet, the timing and mode of administration, the rate of excretion,medicaments used in combination and the severity of the particulardisorder to which the therapy is applied. Dosages for a given patientcan be determined using conventional considerations, e.g., by customarycomparison of the differential activities of the compositions of theinvention and of the therapeutic agent administered alone, such as bymeans of an appropriate conventional pharmacological or prophylacticprotocol.

The maximal dosage for a subject is the highest dosage that does notcause undesirable or intolerable side effects. The number of variablesin regard to an individual prophylactic or treatment regimen is large,and a considerable range of doses is expected. The route ofadministration will also impact the dosage requirements. It isanticipated that dosages of the compositions will reduce symptoms of thecondition being treated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% compared to pre-treatment symptoms or symptoms is leftuntreated. It is specifically contemplated that pharmaceuticalpreparations and compositions may palliate or alleviate symptoms of thedisease without providing a cure, or, in some embodiments, may be usedto cure the disease or disorder.

Suitable effective dosage amounts for administering the compositions maybe determined by those of skill in the art, but typically range fromabout 1 microgram to about 10,000 micrograms per kilogram of body weightweekly, although they are typically about 1,000 micrograms or less perkilogram of body weight weekly. In some embodiments, the effectivedosage amount ranges from about 10 to about 10,000 micrograms perkilogram of body weight weekly. In another embodiment, the effectivedosage amount ranges from about 50 to about 5,000 micrograms perkilogram of body weight weekly. In another embodiment, the effectivedosage amount ranges from about 75 to about 1,000 micrograms perkilogram of body weight weekly. The effective dosage amounts describedherein refer to total amounts administered, that is, if more than onecompound is administered, the effective dosage amounts correspond to thetotal amount administered.

The effectiveness of the compositions described herein may be enhancedby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% relative to acontrol treated with the therapeutic agent alone. It will be appreciatedthat the effectiveness of the treatment in any given case will beenhanced variably in accordance with the specific compositions used, thetype of disease being treated, the condition of the subject, thespecific formulations of the compounds and other relevant medicalfactors that may modify the activity of the compositions or theresponses of the subject as is appreciated by those of skill in the art.

The following examples are meant only to be illustrative and are notmeant as limitations on the scope of the invention or of the appendedclaims. All references cited herein are hereby incorporated by referencein their entireties.

EXAMPLES Example 1: CBD Targets Partially Untwisted or UndertwistedRegions of Collagen

Clostridium histolyticum collagenase causes extensive degradation ofcollagen in the connective tissue resulting in gas gangrene. TheC-terminal collagen-binding domain (CBD) of these enzymes is the minimalsegment required to bind to the collagen fibril. CBD bindsunidirectionally to the partially untwisted C-terminus of triple helicalcollagen. Whether CBD could also target under-twisted regions even inthe middle of the collagen triple helix was examined. Partiallyuntwisted collagenous peptides were synthesized by introducing a Gly→Alasubstitutions into the collagen ([(POG)_(x)POA(POG)_(y)]₃ where x+y=9and x>3). ¹H-¹⁵N heteronuclear single quantum coherence nuclear magneticresonance (HSQC NMR) titration studies with ¹⁵N-labeled CBD demonstratedthat the untwisted mini-collagen binds to a 10 Å wide 25 Å long cleft.Six untwisted collagenous peptides each labeled with a nitroxide radicalwere then titrated with ¹⁵N-labeled CBD. The paramagnetic nuclear spinrelaxation effects showed that CBD binds close to either the Gly→Alasubstitution site or to the C-terminus of each mini-collagen. Smallangle X-ray scattering (SAXS) measurements revealed that CBD prefers tobind the Gly→Ala site rather than the C-terminus. The HSQC NMR spectraof ¹⁵N-labeled mini-collagen and untwisted mini-collagen were unaffectedby the titration of unlabeled CBD. The results imply that CBD binds tothe partially unwound region of the mini-collagen but does not activelyunwind the triple helix.

Materials and Methods:

¹⁵N-Labeled Protein Production:

The s3b (Gly893-Lys1008) peptide derived from Clostridium histolyticumclass I collagenase (ColG) was expressed as a glutathione S-transferase(GST)-fusion protein. The GST-tag was cleaved off by thrombin, and CBDwas purified as described previously. Matsushita, et al., (2001) J BiolChem 276, 8761-8770. Uniform ¹⁵N isotope labeling was achieved usingTanaka minimal medium containing 40 mM ¹⁵NH₄Cl. The labeling efficiencywas estimated to be 99.6% by matrix-assisted laserdesorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).

Peptides:

(POG)₁₀ (SEQ ID NO: 35) was purchased from Peptide Institute, Inc.(Osaka, Japan). Other peptides were constructed by a standardN-(9-fluorenyl) methoxycarbonyl (Fmoc)-based strategy on Rink-amideresins (Novabiochem, Darmstadt, Germany). N-terminal spin-labeling wasperformed on the resin by the treatment with 5 equivalents of3-carboxy-PROXYL (Aldrich), 1-hydroxybenzotriazole,diisopropylcarbodiimide in N,N-dimethylformamide at room temperature for2 hours. Peptide cleavage and de-protection steps were performed by atreatment with a standard trifluoroacetic acid (TFA) scavenger cocktail(TFA: m-cresol:thioanisole:water:triisopropylsilane=82.5:5:5:5:2.5,v/v). The spin-labeling at Cys residues was performed using3-(2-iodoacetamido)-PROXYL (IPSL, Sigma-Aldrich). Briefly, 10 molarexcess of IPSL dissolved in ethanol was added to the same volume of 10mg/ml peptide in 0.1 M Tris-HCl (pH 8.8), 5 mMethylenediaminetetraacetic acid. After reacting at room temperature for1 hr, the reaction was quenched by adding excess dithiothreitol. Allpeptides were purified by reverse-phase HPLC using a Cosmosil 5C₁₈ AR-IIcolumn (Nacalai Tesque, Kyoto, Japan) and characterized by MALDI-TOF-MS.All the measured masses agreed with the expected values. The chemicalstructures of synthesized peptides are shown in FIG. 2.

Circular Dichroism Spectroscopy:

The triple helical conformation and the stability of the collagenouspeptides were verified using CD spectroscopy (See FIGS. 3 and 4). CDspectra were recorded with a J-820 CD spectropolarimeter (JASCO Co.,Hachioji, Japan) equipped with a Peltier thermo controller, using a0.5-mm quartz cuvette and connected to a data station for signalaveraging. All peptide samples were dissolved in water (1 mg/ml), andstored at 4° C. for 24 h. The spectra are reported in terms ofellipticity units per mole of peptide residues [θ]_(mrw).Thermostability of the triple helix was monitored by the [θ]₂₂₅ valuesof each peptide with increasing temperature at the rate of 0.3° C./min.

NMR Spectroscopy:

NMR experiments were performed on a Bruker 700 MHz spectrometer equippedwith Cryoprobe™. All the NMR titration experiments were carried out at16±0.5° C. The working temperature is lower than the meltingtemperatures (T_(M)) of all the paramagnetic spin-labeled collagenouspeptides (Table 1) used. The concentration of the protein was 0.1 mM in50 mM Tris-HCl (pH 7.5) containing 100 mM NaCl and 20 mM CaCl₂. Thedilution effect on the course of titration was minimized by thetitration of a highly concentrated (4 mM) peptide stock. Aliquots ofcollagenous peptide were added to the protein and equilibrated for 5 minbefore acquiring ¹H-¹⁵N HSQC spectra. The pH of the NMR samplesmonitored during the titration exhibited no significant shift in the pH(within ±0.2 units).

TABLE 1 Melting temperatures (T_(m)) of various mini-collagen peptidesthat were used in NMR titration and the experiments described herein.Peptides Tm (° C.) SEQ ID NO: (POG)₄POA(POG)₅ 29 38PROXYL-(POG)₄POA(POG)₅ 29 38 PROXYL-(POG)₃POA(POG)₆ 28 39PROXYL-(POG)₅POA(POG)₄ 28 37 PROXYL-(POG)₆POA(POG)₃ 27 36(POG)₄POA(POG)₅C-PROXYL 30 41 ¹¹PROXYL-(POG)₃PCG(POG)₄ 28 40

Dynamic Light Scattering Experiments:

The dynamic light scattering (DLS) data were collected using DynaPro-Eequipped with a temperature controlled microsampler on the samples ofCBD, collagenous peptides and CBD:mini-collagen complexes in 10 mMTris-HCl (pH 7.5) containing 100 mM sodium chloride and 20 mM CaCl₂. Theprotein samples were spun at 10,000 rpm for 10 min and were filteredthrough 0.02 μm Whatman syringe directly into a 50-μL quartz cuvette.For each experiment, 20 measurements were made. The mean hydrodynamicradius (R_(H)), standard deviation, polydispersity, and percent of peakarea were analyzed using Dynamics V6 (Protein Solutions). Thehydrodynamic radius and molecular weight estimations were calculatedfrom time dependent fluctuations induced by Brownian motion asdescribed. Proteau, et al. (2010) Curr Protoc Protein Sci Chapter 17,Unit 17 10.

Small Angle X-Ray Solution Scattering Experiments:

The small angle X-ray solution scattering (SAXS) data were collected onsolutions of CBD, collagenous peptides and CBD-mini-collagen complexesin 10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 20 mM CaCl₂ at SAXS/WAXSsetup located at the 5-ID-D beamline of the DND-CAT synchrotron researchcenter, Advanced Photon Source, Argonne National Laboratory (Argonne,Ill.). The main advantage of X-ray scattering is that it can be carriedout in solution in near physiological conditions. Petoukhov et al.,(2007) Curr Opin Struct Biol 17, 562-571. 1.2398 Å (10 keV) radiationwas selected from the APS Undulator A insertion device using a Si-111monochromator, with 1:1 horizontal focusing and higher harmonicrejection from a Rh coated mirror, and beam defining slits set at 0.3 mmvertical by 0.25 mm horizontal. A 1.6 mm diameter capillary flow-cellwith a flow rate of 4 μl/sec was used to collect four frames with 10second exposure time. The SAXS detector used was a Mar165 scintillatorfiber-optic coupled CCD detector and covered the momentum transfer range0.005<q<0.198 Å⁻¹, where q=4π sin θ/λ (2θ is the scattering angle). TheWAXS detector was a custom Roper scintillator fiber-optic coupled CCDdetector and covered 0.191<q<1.8 Å⁻¹ S. Weigand, et al. (2009) Advancesin X-ray Analysis 52, 58-68.

All scattering data were acquired at sample temperature of 10° C. Thefour scattering patterns from each detector were averaged and mergedwith the rejection of outlying scans. For further analysis the programIGOR Pro 5.5 A (WaveMetrics) was used. The scattering profiles of theprotein, peptide and their complexes were obtained after subtracting thebuffer profiles. The reduced scattering data were plotted as scatteringintensity I(Q) vs. Q (FIG. 4A). The radius of gyration, R_(g), wasobtained from the Guinier approximation by linear least squares fittingin the QR_(g)<1 region, where the forward scattering intensity I(0) isproportional to the molecular weight of the protein complex. An indirectFourier transformation of I(Q) data using GNOM provided the particledistribution function P(r) in the real space (FIG. 4B). Svergun, D.(1992) J Appl Crystallogr 25, 495-503. Where P(r) intersects with x-axisrepresents the maximum diameter D_(max) averaged in all orientations.The molecular envelopes were constructed for all the samples based onthe SAXS data after ab initio calculation with the program GASBOR.Svergun, et al. (2001) Biophys J 80, 2946-2953. Simulated annealingminimization of randomly distributed dummy atoms converged to theprotein structure after being tested for the best fit to the I(Q)scattering data. No symmetry restraints were applied to any of the shapereconstructions. For each of the complexes, ten ab initio models werecalculated with GASBOR and averaged using DAMAVER. Svergun, D. (2003) JAppl Crystallog 36. The atomic models represented as a compactinterconnected configuration of beads with diameter D_(max) wereadjusted to fit the experimental data I_(exp)(s) to minimize error.Atomic models were docked into ab initio envelopes with the programSUBCOMB. Kozin, M. B., and Svergun, D. (2000) J Appl Crystallogr 33,775-777.

Docking Model:

The CBD-collagenous peptide complex is generated from Protein Data Bankentries of ColG s3b (1NQD) and partially untwisted collagenous peptide1CAG (Ala mutation in 15^(th) position). Other untwisted mini collagenmolecules were generated by modifying 1CAG using fragments derived from[(POG)₁₀]₃ structure (1K6F). To obtain the complex, the soft dockingalgorithm BiGGER was used. Palma, et al. (2000) Proteins 39, 372-384.Solutions were filtered using NMR titration data and the highest scoringmodel that satisfied NMR and SAXS results was chosen. The manualadjustments were aided by the use of MIFit. McRee. (1999) J Struct Biol125, 156-165.

Results and Discussion:

¹H-¹⁵N HSQC NMR Titration-CBD Targeting the Under-Twisted Sites inCollagen:

The untwisted collagenous peptide [(POG)₆POA(POG)₃]₃ (SEQ ID NO: 36)that has Ala in the 21^(st) position from the N-terminus wassynthesized. This peptide was further modified to accommodate aparamagnetic spin label at the N-terminus. ¹H-¹⁵N HSQC NMR titrationswere performed with [PROXYL-(POG)₆POA(POG)₃]₃ (SEQ ID NO: 36) and¹⁵N-labeled CBD at ratios ranging from 0.02:1 to 1.5:1. As demonstratedearlier, a total of eleven residues on the collagen binding interface(S928, W956, G971, K995, Y996, L924, T957, Q972, D974, L991 and V993)either disappeared from the HSQC spectrum or exhibited significantchemical shift perturbation from their original position on the courseof titration. Philominathan, et al. (2009) J Biol Chem 284, 10868-10876.The PROXYL group on the N-terminus of the collagenous peptides can causea distance-dependent line broadening of the NMR signals of CBD duringthe course of titration. In addition to the eleven residues, three moreresidues, V973, G975 and S979 exhibited appreciable line broadening andthese residues eventually disappeared from the ¹H-¹⁵N HSQC spectrum ofCBD (FIGS. 5A and 5B). When the [PROXYL-(POG)₆POA(POG)₃]₃ (SEQ ID NO:36):CBD complex was reduced with ascorbic acid those three residuesreappeared in the ¹H-¹⁵N HSQC spectrum. The disappearance of these threeresidues was consistent with the titration of [PROXYL-G(POG)₇]₃ (SEQ IDNO: 42) (C-terminus is at 22^(nd) position from the N-terminal PROXYL)in our earlier publication. The comparison of the two titration resultsdemonstrates that CBD is targeting the Gly→Ala substituted site. If CBDhad only bound to the C-terminus of [PROXYL-(POG)₆POA(POG)₃]₃ (SEQ IDNO: 36) (C-terminus is at 30^(th) position from the N-terminal PROXYL),we would expect to observe the disappearance of only one residue (V973)at the most, as in the published titration of [PROXYL-G(POG)₇(PRG)]₃(SEQ ID NO: 43). The disappearance of the residues (V973, G975 and S979)located at distal side from the Ca²⁺ binding site (FIG. 5C) confirmedthat CBD binds unidirectionally to untwisted collagen as well. Thecollagen binding surface in CBD is a 10-Å-wide and 25-Å-long cleft. Thewidth of the binding cleft in CBD matches the diameter of the triplehelix and its length could accommodate [(POG)₃]₃ (SEQ ID NO: 44). NMRresults imply that CBD is binding to the under-twisted [(POG)₂POA]₃ (SEQID NO: 45) region of the collagen.

As paramagnetic relaxation enhancement is a distance dependentphenomenon, Gly→Ala substitution made at closer to the N-terminal PROXYLgroup should result in the disappearance of more residues on CBD. PROXYLcontaining collagenous peptides, [PROXYL-(POG)₅POA(POG)₄]₃ (SEQ ID NO:37) (Ala at 18^(th) position from the N-terminal PROXYL),[PROXYL-(POG)₄POA(POG)₅]₃ (SEQ ID NO: 38) (Ala at the 15^(th) positionfrom the PROXYL) and [PROXYL-(POG)₃POA(POG)₆]₃ (SEQ ID NO: 39) (Ala atthe 12^(th) position from the PROXYL) were synthesized. Just as in theprevious titrations, the line broadening effects on the residues of CBDwere analyzed from the changes in the ¹H-¹⁵N HSQC spectrum. The shorterthe distance between Gly→Ala substitution site and the N-terminalPROXYL, more residues in CBD disappeared (FIG. 6 and Table 2). Themagnitude of intensity drop for four amide resonances (Q972, G975, S979and L924) of four different mini-collagen molecules was also thefunction of the distance (FIG. 7). The NMR results are consistent withCBD binding to the [(POG)₂POA]₃ (SEQ ID NO: 45) region in each of thefour under-twisted mini-collagen. The binding constants obtained fromall the NMR titrations were <100 μM indicating a moderate bindingaffinity between CBD and under-twisted mini-collagen.

TABLE 2 Residues that disappear due to the presence of PROXYL either atthe N-terminus. C-terminus or in the middle of the collagenous peptidesequence. Alanine Residues disappeared due to No. Peptides positionPROXYL Blank 1 [(POG)10]3 (SEQ ID NO: 35) PROXYL at N-terminus 2[PROXYL-(POG)6POA(POG)3]3 21 V973, G975, S979 (SEQ ID NO: 36) 3[PROXYL-(POG)5POA(POG)4]3 18 Q972, V973, G975, S979 (SEQ ID NO: 37) 4[PROXYL-(POG)4POA(POG)5]3 15 L946, Q972, V973, G975, S979 (SEQ ID NO:38) L946, G953, Q972, V973, D974, 5 [PROXYL-(POG)3POA(POG)6]3 12 G975,N976, V978, S979 (SEQ ID NO: 39) S906, R929, S997, G998 PROXYL atC-terminus 6 [(POG)4POA(POG)5-PROXYL]3 15 V973, G975, S979 and S906,(SEQ ID NO: 41) R929, S997, G998 PROXYL in the middle 7[11PROXYL-(POG)3PCG(POG)4]3 (SEQ ID NO: 40)

The helical conformation in both the [(POG)₂POA]₃ (SEQ ID NO: 45) andthe C-terminal [(POG)₃]₃ (SEQ ID NO: 44) are similarly under-wound. Thedegree of rotation about the screw axis symmetry that describes theinternal triple helical twist is defined as the helical twist value κ.The κ-value oscillates around an average value of −103° for [(POG)₁₀]₃(SEQ ID NO: 35). Bella (2010) J Struct Biol 170, 377-391. The C-terminusof a mini-collagen is under-twisted (κ value shifts from −103° to −110°)but the N-terminus is usually over-twisted. Collagen peptides withGly→Ala substitution in the center of the peptide sequence still formtriple helices, but with an abrupt under-twisting (K value shifts from−103° to −115°) at the substitution site followed over-twisting to thenorm. Because the [(POG)₂POA]₃ (SEQ ID NO: 45) region is somewhat moreunder-twisted than C-terminal [(POG)₃]₃ (SEQ ID) NO: 44), the formercould be preferentially targeted by CBD than the latter. However, CBDcould still bind to the C-terminus.

CBD Also Targets the C-Terminus of the Under-Twisted Mini-Collagen:

To demonstrate that CBD binds to the C-terminal (POG)₃ (SEQ ID NO: 44)as well, a collagenous peptide [(POG)₄POA(POG)₅-PROXYL]₃ (SEQ ID NO: 38)was synthesized. [(POG)₄POA(POG)₅C-PROXYL]₃ (SEQ ID NO: 41) was titratedwith ¹⁵N-labeled CBD at ratios 0.02:1 to 1.5:1 with increments of 0.02,and the changes in the HSQC spectrum of CBD were monitored. When themini-collagen was bound to the cleft, a total of eleven residues on thecollagen binding interface either line broadened or showed significantchemical shift perturbation as described earlier. Philominathan, et al.(2009) J Biol Chem 284, 10868-10876. Four additional residues S906,R929, S997 and G998 disappeared from the HSQC spectrum due to PROXYL(FIGS. 6 A, B and D). These peaks reappeared upon addition of ascorbicacid. This phenomenon is identical to our previous titration of[GPRG(POG)₇C-PROXYL]₃ (SEQ ID NO: 46) when CBD bound the C-terminus. IfCBD were to bind only to the partially unwound Ala site, we would haveobserved the disappearance of fewer residues. Thus in addition totargeting the (POG)₂POA (SEQ ID NO: 45) region of the collagenouspeptide, CBD also binds to the C-terminal (POG)₃ (SEQ ID NO: 44). Asdescribed, the helical confirmation of both the (POG)₂POA (SEQ ID NO:45) region and the C-terminal (POG)₃ (SEQ ID NO: 44) are similarlyunder-twisted compared to the norm. Bella. (2010) J Struct Biol 170,377-391. Our current explanation for why CBD is targeting theunder-twisted regions is that the partial unwinding positions main-chaincarbonyl groups to favor hydrogen-bonding interactions with the hydroxylgroup of Tyr994. Tyr994 mutation to Phe resulted in 12-fold reduction inbinding to mini-collagen, and the mutation to Ala lost bindingcapability. Wilson, et al. (2003) EMBO J 22, 1743-1752.

To demonstrate CBD's ability to target both the (POG)₂POA (SEQ ID NO:45) region and the C-terminal (POG)₃ (SEQ ID NO: 44) region, acollagenous peptide [11PROXYL-(POG)₃PCG(POG)₄]₃ (SEQ ID NO: 40) modifiedto accommodate PROXYL group in the middle (11^(th) position) wassynthesized. PROXYL group is covalently joined to the cysteine residue.Due to the presence of the bulky PROXYL group, this peptide is expectedto be partially untwisted. The precise degree of under-twisting is notknown for the peptide, but mini-collagen with GPX repeats exhibits amoderate under-twisting (κ=−105°). Bella. (2010) J Struct Biol 170,377-391. The bulky PROXYL group will likely induce greater untwistingthan κ=−105°. In addition to the eleven amide resonances eitherline-broaden or shifted, ¹H-¹⁵N HSQC NMR titrations revealed twodistinct phenomena. At lower ratio (0.2:1) amide resonancescorresponding to S906, R929, S997, and G998 disappeared from the HSQCspectrum of CBD (FIGS. 8E, F and H). Then at higher ratio (0.3:1),additional amide resonances corresponding to V973, G975 and S979disappeared from the HSQC spectrum of CBD (FIGS. 8E, F and H). In orderto cause the disappearance of four residues (S906, R929, S997 and G998),CBD must initially bind to the N-terminal (POG)₃ (SEQ ID NO: 44). Thedisappearance of resonances V973, G975 and S979 can be explained if CBDbinds to the C-terminal (POG)₃ (SEQ ID NO: 44) of the mini-collagen.However the initial phenomenon signifies that CBD binds preferentiallyto the under-twisted mid-section to C-terminus.

To demonstrate that PROXYL caused the line broadening and Ala or Cysresidues did not, three more control peptides, [(POG)₄POA(POG)₅]₃ (SEQID NO: 38), [(POG)₄POA(POG)₅C-carbamidomethyl]₃ (SEQ ID NO: 41), and[(POG)₃PCG(POG)₄]₃ (SEQ ID NO: 40) that lack the PROXYL groups weresynthesized, and NMR titrations were repeated (FIGS. 6F, 8C and 8G,respectively). The titration results were nearly identical with those of[(POG)₁₀]₃ (SEQ ID NO: 35). Only the eleven amide resonances were eitherline broadened or shifted even at 1:1 (mini-collagen:CBD) ratio. Thesecontrol peptides bound to the same cleft, and PROXYL caused theadditional residues to line broaden.

To illustrate if CBD binds only to the partially untwisted site in themiddle of the collagen peptide and/or to the C-terminus ofmini-collagen, dynamic light scattering experiments (DLS) wereperformed. DLS experiments provided the stoichiometries of collagen:CBDcomplexes. The hydrodynamic radius of [(POG)₄POA(POG)₅-PROXYL]₃ (SEQ IDNO: 38):CBD and [11PROXYL-(POG)₃PCG(POG)₄]₃ (SEQ ID NO: 40):CBD was 3 nmand the apparent molecular weight of the complex was 42±1 kDa, which issimilar to those observed for [(POG)₁₀]₃ (SEQ ID NO: 35):CBD complex(Table 3). Other complexes also exhibited similar values. Thus far, allthe mini-collagen and CBD always formed 1:1 complex. CBD binds to eitherone of the available sites in mini-collagen but does not occupy bothsites to form a 1:2 complex.

TABLE 3 Hydrodynamic radius (RH), apparent molecular weight (Mw), Radiusof gyration (Rg) and Maximum particle diameter (Dmax) computed fromDynamic light scattering (DLS) and small angle X-ray scattering (SAXS)for various CBD:collagenous peptides complexes. Dynamic Light Scattering(DLS) Small Angle X-ray Hydro- Apparent Scattering (SAXS) dynamicMolecular Radius of Max Radius Weight Gyration Diameter No Complexes(RH) (Mw) (Rg) (Dmax) 1 CBD:[(POG)10]3 (SEQ ID NO: 35) 3 43 22.62 ± 0.0493 2 CBD:[PROXYL-(POG)6POA(POG)3]3 3 44 24.67 ± 0.09 87 (SEQ ID NO: 36)3 CBD:[PROXYL-(POG)5POA(POG)4]3 3 42 21.08 ± 0.02 90 (SEQ ID NO: 37) 4CBD:[PROXYL-(POG)4POA(POG)5]3 3 43 25.48 ± 0.08 92 (SEQ ID NO: 38)CBD:[(POG)4POA(POG)5]3 3 43 24.45 ± 0.14 85 (SEQ ID NO: 38) 5CBD:[PROXYL-(POG)3POA(POG)6]3 3 42 21.97 ± 0.14 94 (SEQ ID NO: 39) 6CBD:[(POG)4POA(POG)5C-PROXYL]3 3 44 24.09 ± 0.16 85 (SEQ ID NO: 41)CBD:[(POG)4POA(POG)5]3 3 42 24.67 ± 0.1  84 (SEQ ID NO: 38) 7CBD:[11PROXYL-(POG)3PCG(POG)4]3 3 42 96 (SEQ ID NO: 40)CBD:[(POG)3PCG(POG)4]3 3 43 23.59 ± 0.05 90 (SEQ ID NO: 40)

Small Angle X-Ray Scattering Experiments (SAXS):

The three dimensional molecular shapes of the CBD-collagenous peptidecomplexes were constructed using SAXS measurements. The main advantageof SAXS measurements is that the experiments are performed in solutionunder near physiological conditions. In our previous work, these threedimensional molecular envelopes were used to demonstrate asymmetricbinding of CBD to the C-terminal (POG)₃ (SEQ ID NO: 44) ofmini-collagen. The molecular shapes were constructed for complexes ofCBD and six different untwisted mini-collagen molecules. In all casesCBD bound to (POG)₂POA (SEQ ID NO: 45) region preferentially toC-terminal (POG)₃ (SEQ ID NO: 44) (FIGS. 9A-F). For example the dockingmodel for CBD:[(POG)₄POA(POG)₅]₃ (SEQ ID NO: 38) constructed using thecrystal structure of CBD (pdb accession code 1NQD) interacting with(POG)₂POA (SEQ ID NO: 45) region of the untwisted collagen (pdbaccession code 1CAG) fit the envelope well (FIG. 9B). Although NMRresults demonstrated that CBD also binds to the C-terminal (POG)₃ (SEQID NO: 44) of [(POG)₄POA(POG)₅-PROXYL]₃ (SEQ ID NO: 38), CBDpredominantly binds to the (POG)₂POA (SEQ ID NO: 45) region of thepeptide (FIGS. 9E and 9F).

Structures derived from SAXS profiles using simulated annealingcalculations for [11PROXYL-(POG)₃PCG(POG)₄]₃ (SEQ ID NO: 40) (FIGS. 9Gand 9H) indicated an additional density that could be attributed to thePROXYL group. The SAXS derived three-dimensional shape of[11PROXYL-(POG)₃PCG(POG)₄]₃ (SEQ ID NO: 40):CBD complex superimposeswell with either NMR derived complexes i.e., CBD binding to theN-terminal (POG)₃ (SEQ ID NO: 44) or to the C-terminal (POG)₃ (SEQ IDNO: 44) (FIGS. 9G and 9H).

Little Structural Change of ¹⁵N-Minicollagen Upon CBD Binding:

The studies thus far suggest that CBD scans the collagen fibril forunder-twisted regions. Upon binding to the less structured regions, doesit actively unwind collagen? Active unwinding by CBD would facilitatecollagenolysis. To investigate two collagenous peptides selectivelylabeled with 15N near N- or near C-terminus of [(POG)₁₀]₃ (SEQ ID NO:35) were synthesized (Table 4, peptides A, B), and the structuralchanges due to the binding of unlabeled CBD were monitored using ¹H-¹⁵NHSQC titration.

TABLE 4 ¹⁵N-Labeled Mini-collagen SEQ ID NO: APOGPOG*POGPOGPOGPOGPOGPOGPOGPOG 35 B POGPOGPOGPOGPOGPOGPOGPOGPOG*POG 35C POGPOG*POGPOGPOAPOGPOGPOGPOGPOG 38 D POGPOGPOGPOGPOAPOGPOGPOGPOG*POG38 *indicates the ¹⁵N-labeled Glycine; A indicates Gly → Alasubstitution.

The ¹⁵N-Gly labeled peptides exhibited two distinct cross peaks in the¹H-¹⁵N HSQC spectrum (FIGS. 10A and 10B). Those cross peaks correspondedto unwound monomer and triple helical conformations assigned in earlierNMR studies. Liu, et al. (1996) Biochemistry 35, 4306-4313 and Li, etal. (1993) Biochemistry 32, 7377-7387. The Gly residue closer to theterminal triplets exhibits both monomer and trimer peaks in the HSQCspectrum, whereas the Gly residue in the middle of the triple helixexhibits a strong trimer cross peak. If CBD is to bend or to cause anyunwinding of the triple helix upon binding, we expected the cross peakcorresponding to the triple helix to line broaden and disappear on thecourse of titration, and the cross peak corresponding to the singlechain to intensify. However during the course of the titration, CBD didnot instigate any changes on the ¹H-¹⁵N HSQC spectra of the collagenouspeptides. Thus CBD bound to C-terminal (POG)₃ (SEQ ID NO: 35) imposedlittle structural changes to the triple helix.

Untwisted mini-collagen molecule selectively labeled with ¹⁵N-Gly eitherat near the N- or C-termini (Table 4C and D) was titrated with unlabeledCBD. Cross peaks corresponding to monomer and triple-helix wereidentified on the HSQC spectra (FIGS. 10C and 10D). The titration ofunlabeled CBD induced little change in the intensity of either monomeror trimer cross peak. Even upon binding to the partially unwoundmini-collagen, CBD does not initiate any further unwinding.

CBD unidirectionally binds to the under-twisted site in the triplehelical collagen. CBD may help disband the collagen fibril, but does notunwind the triple helix. Targeting under-twisted regions oftropocollagen may circumvent the energy barrier required for unwindingthe triple helices. When CBD is used as a drug delivery molecule, theinjected molecule distributes prominently to the end plates of vertebraldiscs, near the growth plates of tibia and fibula, and also to skin. Itcould be unloading its payload to the most blood accessible collagenthat is undergoing remodeling, thus rich in under-twisted regions.

Example 2: Structural Comparison of ColH and ColG Collagen-BindingDomains

The C-terminal collagen-binding domain (CBD) of collagenase is requiredfor insoluble collagen fibril binding and for subsequent collagenolysis.The high resolution crystal structures of ColG-CBD (s3b) and ColH-CBD(s3) the molecules resemble one another closely (r.m.s.d. C_(α)=1.5 Å),despite sharing only 30% sequence identity. Five out of six residueschelating Ca²⁺ are conserved. The dual Ca²⁺ binding sites in s3 arecompleted by a functionally equivalent aspartate. The three mostcritical residues for collagen interaction in s3b are conserved in s3.The general shape of the binding pocket is retained by altered loopstructures and side-chain positions. Small angle X-ray scattering datarevealed that s3 also binds asymmetrically to mini-collagen. Besides thecalcium-binding sites and the collagen-binding pocket, architecturallyimportant hydrophobic residues and hydrogen-bonding network around thecis-peptide bond are well-conserved in metallopeptidase subfamily M9B.

Common structural features described above and in Bauer et al. (2012) JBacteriol November 9 (which is incorporated herein by reference in itsentirety), enabled us to update the sequence alignment of the CBD in theM9B subfamily (FIG. 1). Conserved residues are important for one of fourreasons: calcium chelation (red), cis-trans isomerization of the linker(yellow), collagen-binding (blue) or protein folding (green). FIG. 1also indicates the strands of the structure along the top of the figure.

The dual calcium-binding site is formed by four chelating residues(Glu899, Glu901, Asn903, and Asp904) within the N-terminal linker, twochelating residues (Asp927 and Asp930) from the β-strand C and invariantTyr1002 hydrogen-bonds and orients Asp930. Residue numbers used in thisparagraph are of s3b. Likewise other supporting cast such as Gly921 isconserved in the middle of β-strand strategically placed to make roomfor Glu899. The dual calcium chelation site is fashioned sometimes byfunctionally equivalent residues. As mentioned, Asp897 of s3 actsequivalently to Asp927 of s3b. Asp897 equivalents are tentativelyidentified in B. brevis s3a and s3b, C. botulinum A3 s3a and C.histolyticum ColG s3a. Tridentate and divalent Asp and Glu residues areconserved with only C. sordellii s3a as the exception. The monodentateAsp904 residue is sometimes substituted by Asn. For those substituted,the net charge of the dual calcium site is neutral rather than −1.

The peptide between residues 901-902 has cis conformation in the holostate for both s3b and s3. The position 902 in other CBD molecules isPro, Asp or Asn. Pro frequently succeeds the peptide bond to easetrans-cis isomerization. The s3 molecule has Pro. In s3b, OD of Asn902hydrogen-bonds with the main-chain N of Asp904. The hydrogen-bond iscritical for the peptide isomerization. Spiriti and van der Vaart.(2010) Biochemistry 49:5314-5320, which is incorporated herein byreference in its entirety. For the remainder of CBD molecules with Aspat the position, OD of Asp could play the same role as that of Asn902.Other hydrogen-bonds identified by simulation studies important instabilizing the transition states are well conserved. Thesedonor-acceptor pairs in s3 and s3b are tabulated (Table 5). Calcium ionscould catalyze the isomerization in all the CBD molecules and theirtransition states and catalytic mechanism may look very similar.

TABLE 5 Hydrogen-bonds important in trans-cis peptide isomerization ins3b and their counterparts in s3. Important H-bonds in s3b fortransition state formation Corresponding H-bonds in s3 T910_OG1 . . .N903_NH2 S879_OG1 . . . N872_ND2 T910_OG1 . . . N900_N S879_OG1 . . .K86_N E899_OE1 . . . N903_ND2 E868_OE1 . . . N872_ND2 E899_OE2 . . .S922_N E868_OE2 . . . T891_N N902_OD1 . . . D904_N NA (N902 replacedwith P871) D930_OD2 . . . Y1002_OH D939_OD2 . . . Y97_OH Y1002_OH . . .Y932_OH NA (Y932 replaced with F901)

Non-functional residues that are important in either folding orarchitectural stability are conserved. Hydrophobic residues packedbetween the Pβ-sheets are better conserved if they are located in thevicinity of functionally critical residues. For example, invariantTrp956 of strand E is packed between the Pβ-sheets. The residuesflanking (Thr955 & Thr957) interact with mini-collagen. Tyr932 is packedbetween the sheets and helps positioning Tyr1002. Residues at tightturns are conserved as well. Gly975 is well conserved to allow a typeII′ turn in s3b. Gly942 (Gly975 equivalent) in s3 allows Asp941side-chain to stabilize the reverse turn. A highly conserved six-residuestretch, between residues 986 and 991, adopts a tight turn and precedesthe functionally important strand H. The region is well ordered in thecrystal structures with low B-factors, and is the least dynamic based onNMR and limited proteolysis MALDI-TOF MS (25). Philominathan, et al.(2009) J Biol Chem 284:10868-10876 and Sides et al. J Am Soc MassSpectrom. (2012) 23(3):505-19 both of which are incorporated herein byreference in their entireties. The main-chain carbonyl and amino groupsof Arg985 hydrogen-bond with OH of Tyr989 to stabilize the turn. OnlyGly987 can make room for the bulky Tyr989 side chain. Tyr990 packsagainst the invariant Ala909 and conserved 3₁₀ helix.Ala909 is at thebase of the linker that undergoes α-helix→β-strand transformation. Thetight turn may ensure that collagen interacting Leu992, Tyr994, andTyr996 would be correctly positioned. Tyr994 is the most criticalresidue in interacting with collagenous peptides. Wilson, et al. (2003)EMBO J 22:1743-1752. The strands adjacent to strand H, i.e. strands Cand E, are very well conserved. The three antiparallel strands mold thecollagen-binding pocket. Strand F staples the Pβ-sheets by interactingwith both sheets. The β-strand first interacts in an antiparallelorientation with strand E then breaks its direction at Gly971 tointeract with strand G. In place of Gly971, Ala or Pro is found at thelocation where the strand switches its allegiance. The dual interactionof the strand helps positioning Tyr970 to interact with mini-collagen.

Three residues shown to interact strongly with mini-collagen areconserved. The invariant Tyr994 and well conserved Tyr970 and Tyr996constitute the “hot spot”. Y994A mutation lost binding capability. SinceY994F resulted in 12-fold reduction in binding to mini-collagen, thehydroxyl group of Tyr994 may interact with collagen through ahydrogen-bond. Tyr996, which is a critical residue in bindingmini-collagen, is not so well conserved. Y996A caused 40-fold reductionin binding to the mini-collagen. Y996 is s3b is replaced with Phe in s3,though both side chains have identical orientation. In other CBDmolecules, an aromatic residue, such as Phe or His, is sometimes foundat the site. Y970A results in 12-fold reduction in binding tomini-collagen. Thr957 was found to interact with mini-collagen by¹⁵N-HSQC-NMR titration. The β-branched amino acid residues or Leu arefound at the positions equivalent to Thr957 in most of the CBDs. Sixother residues were identified by ¹⁵N-HSQC-NMR titration to interactwith mini-collagen are not very well conserved. Since divergent CBDs (s3and s3b) adopted a similar saddle-shaped binding pocket, other CBDs mayalso adopt similar collagen-binding strategy.

Divergent CBD could target different collagen sequences and couldpossibly target different collagen types; however, this structural studysuggest otherwise. Rather, all the CBD domains may bind similarly to anunder-twisted region such as the C-terminus of a collagen fibril. TheC-terminus of type I collagen is exposed in the fibril surface based onX-ray fiber diffraction experiments, and it is the most accessible sitefor the bacterial collagenase to initiate assaults. However CBD bindingonly at the C-terminal region of tropocollagen is unfounded. Goldparticle-labeled tandem ColG-CBD (s3a-s3b) labeled with gold particlebound to type I collagen fibrils exhibited no periodicity. In thecollagen fibrils, the molecules are staggered from each other by about67 nm. Therefore CBD could target partially under-twisted regions in themiddle of a tropocollagen that are also vulnerable for assaults.

Much like s3b, s3 is both compact, and extremely stable in the presenceof physiological Ca²⁺. Thus, the enzyme could degrade extracellularmatrix for prolonged time. The linker that induced structuraltransformation is a common feature found in M9B collagenase. It couldact as Ca²⁺ sensor to trigger domain rearrangement as means of enzymeactivation. Ca²⁺ concentration in extracellular matrix is higher thanthat inside a bacterium. Both s3 and s3b bind similarly to amini-collagen, thus M9B collagenase molecules could initiatecollagenolysis from analogous structural features in various collagenfibril. Fusion protein of any CBD derived from M9B collagenase and agrowth factor should result in comparable clinical outcome.

Example 3: CBD-PTH Agonist Spurs Hair Growth and CBD-PTH AntagonistInhibits Hair Growth

In-Vitro Characterization of CBD-Linked PTH Compounds:

Collagen binding of each peptide was verified in flow-through collagenbinding assays as previously described in U.S. Patent Publication No.2010/0129341, which is incorporated herein by reference in its entirety.PTH-CBD, consisting of the first 33 amino acids of PTH linked directlyto the collagen binding domain (SEQ ID NO: 1), was the most potentagonist, having a similar effect to that of PTH(1-34) (SEQ ID NO: 7) oncAMP accumulation. Ponnapakkam et al. (2011) Calcif 88:511-520. Epub 211April 2022. Among the antagonists, PTH(7-33)-CBD (SEQ ID NO: 10) had thebest combination of low intrinsic activity and high receptor blockade(not shown), similar to those seen in other PTH antagonists, includingthose used in hair growth studies. Peters, et al. (2001) J InvestDermatol 117:173-178.

In-Vivo Distribution of PTH-CBD:

Tissue distribution was assessed by administering ³⁵S-labelled PTH-CBDvia subcutaneous injection, followed by whole mount frozen andwhole-body autoradiography. PTH-CBD with a phosphorylation site betweenPTH(1-33) and the CBD was purified, activated and labeled with[gamma-35] ATP as described previously. Tamai et al. (2003) InfectImmun. 71:5371-5375. Approximately 10.8 mcg of ³⁵S-PTH-CBD (122 kcm/mcg)was injected subcutaneously in 7 week-old mice (32-35 g). Mice weresacrificed at 1 hour or 12 hours post-injection, and then frozen in dryice-acetone. Frozen sections (50 am) were prepared with an autocryotome,dried at −20° C., and exposed to an image plate for 4 weeks. Thereappeared to be an initial distribution of ³⁵S-PTH-CBD to a broad area ofskin around the site of injection, followed by a rapid redistribution tothe skin of the entire animal, as well as to several other tissues (i.e.bone, intestine, bladder) (FIG. 11). PTH-CBD thus showed the desiredproperties of distribution and retention to skin with subcutaneousadministration.

PTH-CBD Reverses Hair Loss in Chemotherapy-Induced Alopecia in Mice:

We compared efficacy of CBD linked PTH agonists and antagonists inchemotherapy-induced alopecia, utilizing an experimental designpublished by Peters, et al., for non-CBD linked PTH compounds. Peters,et al. (2001) J Invest Dermatol 117:173-178. C57BL/6J mice (JacksonLaboratories, Bar Harbor, Me.) were depilated to synchronize the hairfollicles, and cyclophosphamide (CYP, 150 mg/kg) was administered on day9 to maximize the chemotherapy-induced damage. The agonist (PTH-CBD) andthe antagonist (PTH(7-33)-CBD) were administered 2 days prior tochemotherapy, and given the long-term retention of the compounds in theskin, we administered only a single dose to cover the timing of themultiple injections of PTH agonist and antagonist in the study byPeters, et. al. The administered dose of CBD-linked compounds (320mcg/kg) is well tolerated in mice. Ponnapakkam et al. (2011) Calcif88:511-520. Epub 211 April 2022.

The results of the photodocumentation record indicate that the agonist,PTH-CBD, was far more effective at stimulating hair growth than was theantagonist (FIG. 12). Histological examination revealed morphologicalchanges in the hair follicles after CYP therapy, which were moresuperficially located and exhibited clumped melanocytes around the bulb,characteristics of the dystrophic anagen and catagen phase (FIG. 13).While the antagonist PTH(7-33)-CBD had no beneficial effect, treatmentwith the agonist PTH-CBD led to deeper rooting and reduced melanocyteclumping, thus reversing the dystrophic changes. Counts of anagen VIhair follicles per high-powered field (HPF) were compared betweengroups; animals treated with PTH-CBD had a higher number of hairfollicles, approaching those of animals which did not receivechemotherapy (FIG. 14), while the antagonist PTH(7-33)-CBD had nobeneficial effect.

Importantly, we saw no evidence of adverse effects from PTH-CBDadministration. While PTH injections are known to elevate blood calciumand can cause kidney stones, PTH-CBD had no effect on serum calcium. Inaddition, there was no evidence of excess hair length on the body or ofexcess hair growth on the ears and tail, where a full coat is normallynot present. The effects of PTH-CBD on hair growth have been confirmedin models of chemotherapy-induced alopecia without depilation, whichmore closely mimic clinical protocols.

Quantification of Effects of PTH-CBD in Chemotherapy-Induced Alopecia:

We followed these studies by comparing the effects of different doses ofPTH-CBD in chemotherapy-induced alopecia. In these studies, we appliedthe injections more distally on the back and applied a gray-scaleanalysis to quantify the amount of hair growth. Injecting more distallyin the back allows us to compare regrowth of hair after PTH-CBDtreatment with less interference from the normal hair regrowth, whichnormally proceeds from head to tail in mice. The results are shown inFIG. 15, indicating a dose-dependent effect on hair regrowth bothqualitatively and quantitatively.

Chemotherapy-Induced Alopecia without Depilation:

While the depilated model of chemotherapy-induced alopecia provides auniform model for comparison of drug effects, the depilation process isknown to cause hair follicle injury, and may alter the response of theanimals to the PTH-CBD administration. We therefore tested the effectsof PTH-CBD in another model of chemotherapy-induced alopecia, where theanimals were given 3 courses of cyclophosphamide therapy (50 mg/kg/wk),similar to the usual manner in which cancer patients might be treated.In this model, it takes much longer (4-6 months) for alopecia todevelop. Animals that received a single dose of PTH-CBD (320 mcg/kgsubcutaneous) prior to the first cycle did not develop hair loss asshown in FIG. 16.

In a second study, we compared the effects of PTH-CBD when givenprophylactically, at the time of the first cycle of chemotherapy, vs.therapeutically, after the hair loss had developed. While PTH-CBD waseffective in both instances, the effects were more prominent when givenprophylactically. This is evident both visually and quantitatively inFIG. 17, using the same grey scale analysis used in our dose-responsestudy.

Depilation Alopecia:

The agonist PTH-CBD appears to increase hair growth by increasing thenumber of anagen phase hair follicles. As such, there is no reason tobelieve that hair growth effects should be limited to the chemotherapymodel. We therefore tested both PTH-CBD and antagonist compound,PTH(7-33)-CBD, after removing hair from C57/BL6J mice by waxing (FIG.18). The results were quite interesting; agonist (PTH-CBD) treatedanimals had earlier anagen eruption (day 7 vs. day 9 for vehiclecontrols), and exhibited more complete regrowth of hair by the end ofthe study (day 18). Antagonist (PTH(7-33)-CBD) treated animals also hadan early anagen eruption, but the hair growth which followed wasmarkedly curtailed, and the hair cycle was arrested after this point,resulting no further observed regrowth of hair. Thus, it appears thatagonist therapy is acting to promote more rapid regrowth of hair bypromoting more rapid transition to the anagen phase, while theantagonist inhibited hair regrowth by blocking this transition.

PTH-CBD is a fusion protein of the first 33 amino acids of parathyroidhormone (PTH) and a bacterial collagen binding domain. The collagenbinding activity causes PTH-CBD to be retained at its site of action inthe dermal collagen, maximizing efficacy and reducing systemicside-effects. PTH-CBD stimulates hair growth by causing hair folliclesto enter an anagen VI or growth phase, presumably by activating WNTsignaling and increasing production of beta-catenin. We therefore planto conduct the following additional studies to confirm this mechanism ofaction and to determine the effect of PTH-CBD in two distinct geneticmouse models with WNT signaling inhibition. These data will be used informulating clinical trials for PTH-CBD as a therapy for alopecia.

Alopecia Areata:

Alopecia Areata is a disease of patchy hair loss due to autoimmunedestruction of the hair follicles. We tested the efficacy of PTH-CBD inpromoting regrowth of hair in an animal model of alopecia areata, theengrafted C3H/Hej mouse. In this model, hair loss develops variably overthe first 2 months of life. Shown in FIG. 19 is the results of a singledose of PTH-CBD (320 mcg/kg subcutaneous) administered into theengrafted site, the center of the back, where there was maximal hairloss. Compared to vehicle control animals, which continued to lose hairat this site, animals receiving PTH-CBD began to show regrowth of hairwithin the next 1-4 days. Importantly, the response was found to besustained during the 2 month course of the experiment.

Example 4: CBD-PTH can Prevent or Treat Hyperparathyroidism

In this experiment, rats had their ovaries surgically removed at age 3months. At age 9 months, rats were injected with either a single dose ofPTH-CBD (320 mcg/kg) or vehicle control. Animals were sacrificed 6months after therapy (age 15 months). Human intact PTH levels weremeasured to assess serum levels of PTH-CBD, and were found to beundetectable in both groups. Serum calcium was measured and there wereno differences between groups (Vehicle: 13.5+/−1.1 vs. PTH-CBD:14.3+/−1.1 mg/dl, NS). Rat intact PTH levels were measured to assessendogenous PTH production, and PTH-CBD suppressed the normal increase inendogenous PTH levels seen in aged, ovarectomized rats. These findingsindicate that a single injection of PTH-CBD can provide long-termsuppression of endogenous PTH production, preventing the normal riseseen with age in the ovarectomized rat model, and thus may serve as atherapy for hyperparathyroidism.

We claim:
 1. A method of delivering a therapeutic agent comprising: (a)selecting a subject in need of treatment for a disease selected from thegroup consisting of osteogenesis imperfecta, Stickler's syndrome,Ehlers-Danlos syndrome, Alport's syndrome, and Caffey's disease, and (b)administering a composition comprising a bacterial collagen-bindingpolypeptide segment linked to a therapeutic agent to the subject,wherein the therapeutic agent is not a PTH/PTHrP receptor agonist orantagonist, and wherein the bacterial collagen-binding polypeptidesegment binds to sites of partially untwisted or under-twisted collagenand delivers the agent thereto, and wherein the bacterialcollagen-binding polypeptide segment comprises a collagen-bindingpolypeptide derived from an M9 peptidase selected from the groupconsisting of Clostridium, Bacillus and Vibrio, one of SEQ ID NOs: 6,13-34, a fragment of at least 8 consecutive amino acids of one of SEQ IDNOs: 6, 13-34, residues 34-158 of SEQ ID NO: 1, a fragment of at least 8consecutive amino acids from residues 34-158 of SEQ ID NO: 1, a peptidethat is at least 90% identical to residues 34-158 of SEQ ID NO: 1, and apeptide that is at least 90% identical to one of SEQ ID NOs: 13-34. 2.The method of claim 1, wherein the therapeutic agent is an agent capableof promoting bone growth, decreasing inflammation, or promoting collagenstability.
 3. The method of claim 1, wherein the therapeutic agent isselected from the group consisting of BMP-2, BMP-3, FGF-2, FGF-4,anti-sclerostin antibody, growth hormone, IGF-1, VEGF, TGF-β, KGF,FGF-10, TGF-α, TGF-β1, TGF-β receptor, GM-CSF, EGF, PDGF and connectivetissue growth factors.
 4. The method of claim 1, wherein the bacterialcollagen-binding polypeptide segment comprises a collagen-bindingpolypeptide selected from the group consisting of residues 34-158 of SEQID NO: 1, a fragment of at least 8 consecutive amino acids from residues34-158 of SEQ ID NO: 1, and a peptide that is at least 90% identical toresidues 34-158 of SEQ ID NO:
 1. 5. The method of claim 1, wherein thecollagen-binding polypeptide segment and the therapeutic agent arechemically cross-linked to each other or are polypeptide portions of afusion protein.
 6. The method of claim 1, wherein the therapeutic agentis a polypeptide and the N-terminus of the collagen-binding polypeptidesegment is linked directly or through a linker polypeptide segment tothe C-terminus of the therapeutic agent polypeptide.
 7. The method ofclaim 1, wherein the composition has at least 50% greater activity inthe subject than the therapeutic agent administered alone.
 8. The methodof claim 1, wherein the composition is administered intramuscularly,intradermally, intravenously, subcutaneously, intraperitoneally,topically, orally, parenteral, or intranasally.
 9. The method of claim1, wherein the subject is a human.
 10. The method of claim 1, whereinthe composition is administered in aqueous solution at pH below about5.0 or above about 6.0.
 11. The method of claim 6, wherein the linkerpolypeptide includes a polycystic kidney disease (PKD) domain of thecollagen-binding protein.
 12. The method of claim 11, wherein the PKDdomain comprises residues 807-901 of SEQ ID NO:
 6. 13. The method ofclaim 1, wherein the collagen-binding polypeptide includes residues894-1008, 894-1021, 901-1021, or 901-1008 of SEQ ID NO: 6 or a homologthereof.
 14. The method of claim 6, wherein collagen binding polypeptideinclude residues 37-251 of SEQ ID NO: 2 or residues 807-1021 of SEQ IDNO:
 6. 15. The method of claim 1, wherein the collagen bindingpolypeptide comprises residues 34-158 of SEQ ID NO:
 1. 16. The method ofclaim 1, wherein the collagen binding polypeptide comprises a peptidethat is at least 90% identical to one of SEQ ID NOs: 13-34.
 17. Themethod of claim 1, wherein the collagen binding polypeptide is a peptidethat is at least 90% identical to residues 34-158 of SEQ ID NO: 1.